The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

Poor quality Vß recombination signal sequences stochastically enforce TCRß allelic exclusion.

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vß gene segments with a higher quality target elevates Vß rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRß chains derived from both Tcrb alleles. Thus, TCRß allelic exclusion is enforced genetically by the low quality of Vß recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.

V(D)J Recombination Exploits DNA Damage Responses to Promote Immunity.

It has been recognized for 40 years that the variable (diversity) joining [V(D)J] recombination-mediated assembly of diverse B and T lymphocyte antigen receptor (AgR) genes is not only essential for adaptive immunity, but also a risk for autoimmunity and lymphoid malignancies. Over the past few years, several studies have revealed that recombination-activating gene (RAG) endonuclease-induced DNA double-strand breaks (DSBs) transcend hazardous intermediates during antigen receptor gene assembly. RAG cleavage within the genomes of lymphocyte progenitors and immature lymphocytes regulates the expression of ubiquitous and lymphocyte-specific gene transcripts to control the differentiation and function of both adaptive and innate immune cell lineages. These unexpected discoveries raise important new questions that have broad implications for basic immunology research and the screening, diagnosis, and treatment of human immunological disease.

Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks.

Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ataxia telangiectasia mutated (ATM) kinase to feedback-inhibit RAG expression and RAG cleavage of the other Igκ allele. In this article, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA, but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic Ag receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of monospecific lymphocytes while suppressing oncogenic Ag receptor locus translocations.

Lymphocyte lineage-specific and developmental stage specific mechanisms suppress cyclin D3 expression in response to DNA double strand breaks.

Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs. This response requires the ATM protein kinase that is activated by DSBs. Cyclin D3 protein loss in thymocytes coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that thymocytes repress Cyclin D3 expression via ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene represses Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from suppressing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T cells use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit Cyclin D3 protein levels and thereby help prevent cellular proliferation in response to DSBs. We discuss the relevance of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

UNLABELLED: The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. IMPLICATIONS: A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis.

Genomic Alterations of Non-Coding Regions Underlie Human Cancer: Lessons from T-ALL.

It has been appreciated for decades that somatic genomic alterations that change coding sequences of proto-oncogenes, translocate enhancers/promoters near proto-oncogenes, or create fusion oncogenes can drive cancer by inducing oncogenic activities. An explosion of genome-wide technologies over the past decade has fueled discoveries of the roles of three-dimensional chromosome structure and powerful cis-acting elements (super-enhancers) in regulating gene transcription. In recent years, studies of human T cell acute lymphoblastic leukemia (T-ALL) using genome-wide technologies have provided paradigms for how non-coding genomic region alterations can disrupt 3D chromosome architecture or establish super-enhancers to activate oncogenic transcription of proto-oncogenes. These studies raise important issues to consider with the objective of leveraging basic knowledge into new diagnostic and therapeutic opportunities for cancer patients.

Regulation of Tcrb Gene Assembly by Genetic, Epigenetic, and Topological Mechanisms.

The adaptive immune system endows mammals with an ability to recognize nearly any foreign invader through antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assemble antigen receptor genes from physically separated variable (V), joining (J), and sometimes diversity (D) gene segments. The recombination process itself must be stringently regulated to minimize oncogenic translocations involving chromosomes that harbor immunoglobulin and T cell receptor loci. Indeed, V(D)J recombination is controlled at several levels, including tissue-, developmental stage-, allele-, and gene segment-specificity. These levels of control are imposed by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Together, the genetic elements regulate developmental changes in chromatin, transcription, and locus topology that promote or disfavor long-range recombination. This chapter focuses on the cross talk between these mechanisms at the T cell receptor beta (Tcrb) locus, and how they sculpt a diverse TCRß repertoire while maintaining monospecificity of this antigen receptor on each mature T lymphocyte. We also discuss how insights obtained from studies of Tcrb are more generally relevant to our understanding of gene regulation strategies employed by mammals.

B Cell-Intrinsic Expression of the HuR RNA-Binding Protein Is Required for the T Cell-Dependent Immune Response In Vivo.

The HuR RNA-binding protein posttranscriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted Abs, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all Ab isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity Abs after immunization with a T cell-dependent Ag. This failure of HuRΔ/Δ mice to mount a T cell-dependent Ab response contrasts with the ability of HuRΔ/Δ B cells to become GC-like in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of costimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T cell-dependent immune responses in vivo.

Somatic inactivation of ATM in hematopoietic cells predisposes mice to cyclin D3 dependent T cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of immature T cells that exhibits heterogeneity of oncogenic lesions, providing an obstacle for development of more effective and less toxic therapies. Inherited deficiency of ATM, a regulator of the cellular DNA damage response, predisposes young humans and mice to T-ALLs with clonal chromosome translocations. While acquired ATM mutation or deletion occurs in pediatric T-ALLs, the role of somatic ATM alterations in T-ALL pathogenesis remains unknown. We demonstrate here that somatic Atm inactivation in haematopoietic cells starting as these cells differentiate in utero predisposes mice to T-ALL at similar young ages and harboring analogous translocations as germline Atm-deficient mice. However, some T-ALLs from haematopoietic cell specific deletion of Atm were of more mature thymocytes, revealing that the developmental timing and celluar origin of Atm inactivation influences the phenotype of ATM-deficient T-ALLs. Although it has been hypothesized that ATM suppresses cancer by preventing deletion and inactivation of TP53, we find that Atm inhibits T-ALL independent of Tp53 deletion. Finally, we demonstrate that the Cyclin D3 protein that drives immature T cell proliferation is essential for transformation of Atm-deficient thymocytes. Our study establishes a pre-clinical model for pediatric T-ALLs with acquired ATM inactivation and identifies the cell cycle machinery as a therapeutic target for this aggressive childhood T-ALL subtype.

Tcrδ translocations that delete the Bcl11b haploinsufficient tumor suppressor gene promote atm-deficient T cell acute lymphoblastic leukemia.

ATM is the master regulator of the cellular response to DNA double strand breaks (DSBs). Deficiency of ATM predisposes humans and mice to αß T lymphoid cancers with clonal translocations between the T cell receptor (TCR) α/δ locus and a 450 kb region of synteny on human chromosome 14 and mouse chromosome 12. While these translocations target and activate the TCL1 oncogene at 14q32 to cause T cell pro-lymphocytic leukemia (T-PLL), the TCRα/δ;14q32 translocations in ATM-deficient T cell acute lymphoblastic leukemia (T-ALL) have not been characterized and their role in cancer pathogenesis remains unknown. The corresponding lesion in Atm-deficient mouse T-ALLs is a chromosome t(12;14) translocation with Tcrδ genes fused to sequences on chromosome 12; although these translocations do not activate Tcl1, they delete the Bcl11b haploinsufficient tumor suppressor gene. To assess whether Tcrδ translocations that inactivate one copy of Bcl11b promote transformation of Atm-deficient cells, we analyzed Atm(-/-) mice with mono-allelic Bcl11b deletion initiating in thymocytes concomitant with Tcrδ recombination. Inactivation of one Bcl11b copy had no effect on the predisposition of Atm(-/-) mice to clonal T-ALLs. Yet, none of these T-ALLs had a clonal chromosome t(12;14) translocation that deleted Bcl11b indicating that Tcrδ translocations that inactivate a copy of Bcl11b promote transformation of Atm-deficient thymocytes. Our data demonstrate that antigen receptor locus translocations can cause cancer by deleting a tumor suppressor gene. We discuss the implications of these findings for the etiology and therapy of T-ALLs associated with ATM deficiency and TCRα/δ translocations targeting the 14q32 cytogenetic region.

The microRNA biogenesis machinery modulates lineage commitment during αß T cell development.

Differentiation of CD4(+) helper and CD8(+) cytotoxic αß T cells from CD4(+)CD8(+) thymocytes involves upregulation of lineage-specifying transcription factors and transcriptional silencing of CD8 or CD4 coreceptors, respectively, in MHC class II or I (MHCII or I)-restricted thymocytes. In this study, we demonstrate that inactivation of the Dicer RNA endonuclease in murine thymocytes impairs initiation of Cd4 and Cd8 silencing, leading to development of positively selected MHCI- and MHCII-restricted mature CD4(+)CD8(+) thymocytes. Expression of the antiapoptotic BCL2 protein or inactivation of the p53 proapoptotic protein rescues these thymocytes from apoptosis, increasing their frequency and permitting accumulation of CD4(+)CD8(+) αß T cells in the periphery. Dicer-deficient MHCI-restricted αß T cells fail to normally silence Cd4 and display impaired induction of the CD8 lineage-specifying transcription factor Runx3, whereas Dicer-deficient MHCII-restricted αß T cells show impaired Cd8 silencing and impaired induction of the CD4 lineage-specifying transcription factor Thpok. Finally, we show that the Drosha RNA endonuclease, which functions upstream of Dicer in microRNA biogenesis, also regulates Cd4 and Cd8 silencing. Our data demonstrate a previously dismissed function for the microRNA biogenesis machinery in regulating expression of lineage-specifying transcription factors and silencing of Cd4 and Cd8 during αß T cell differentiation.

The ataxia telangiectasia mutated and cyclin D3 proteins cooperate to help enforce TCRß and IgH allelic exclusion.

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRß and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRß and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRß or IgH loci in mice lacking ATM. A preassembled DJß complex that decreases the number of TCRß rearrangements needed for a productive TCRß gene further increased frequencies of ATM-deficient cells with biallelic TCRß expression. IgH and TCRß proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRß genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRß or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRß allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.

Noncore RAG1 regions promote Vß rearrangements and αß T cell development by overcoming inherent inefficiency of Vß recombination signal sequences.

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRß and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vß/VH repertoire. Because Vßs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRß gene, but not a preassembled DßJß complex or the prosurvival BCL2 protein, completely rescues αß T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vß utilization in Vß-to-DJß rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vß repertoire during αß TCR selection. Inefficient Vß/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vß14 RSS with a more efficient RSS increases Vß14 recombination and rescues αß T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vß RSS inefficiency to promote Vß recombination and αß T cell development, and by modulating TCRß and TCRα gene segment utilization.

Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations.

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53(-/-) mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53(-/-) mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53(-/-) mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.

Histone H2AX suppresses translocations in lymphomas of Eµ-c-Myc transgenic mice that contain a germline amplicon of tumor-promoting genes.

The DNA damage response (DDR) can restrain the ability of oncogenes to cause genomic instability and drive malignant transformation. The gene encoding the histone H2AX DDR factor maps to 11q23, a region frequently altered in human cancers. Since H2ax functions as a haploinsufficient suppressor of B lineage lymphomas with c-Myc amplification and/or translocation, we determined the impact of H2ax expression on the ability of deregulated c-Myc expression to cause genomic instability and drive transformation of B cells. Neither H2ax deficiency nor haploinsufficiency affected the rate of mortality of Eµ-c-Myc mice from B lineage lymphomas with genomic deletions and amplifications. Yet H2ax functioned in a dosage-dependent manner to prevent unbalanced translocations in Eµ-c-Myc tumors, demonstrating that H2ax functions in a haploinsufficient manner to suppress allelic imbalances and limit molecular heterogeneity within and among Eµ-c-Myc lymphomas. Regardless of H2ax copy number, all Eµ-c-Myc tumors contained identical amplification of chromosome 19 sequences spanning 20 genes. Many of these genes encode proteins with tumor-promoting activities, including Cd274, which encodes the PD-L1 programmed death ligand that induces T cell apoptosis and enables cancer cells to escape immune surveillance. This amplicon was in non-malignant B and T cells and non-lymphoid cells, linked to the Eµ-c-Myc transgene, and associated with overexpression of PD-L1 on non-malignant B cells. Our data demonstrate that, in addition to deregulated c-Myc expression, non-malignant B lineage lymphocytes of Eµ-c-Myc transgenic mice may have constitutive amplification and increased expression of other tumor-promoting genes.

Conditional inactivation of p53 in mature B cells promotes generation of nongerminal center-derived B-cell lymphomas.

The p53 tumor suppressor exerts a central role in protecting cells from oncogenic transformation. Accordingly, the p53 gene is mutated in a large number of human cancers. In mice, germ-line inactivation of p53 confers strong predisposition to development of different types of malignancies, but the early onset of thymic lymphomas in the majority of the animals prevents detailed studies of tumorigenesis in other tissues. Here, we use the Cre/Lox approach to inactivate p53 in mature B cells in mice (referred to as "CP" B cells) and find that such p53 inactivation results in the routine development of IgM-positive CP peripheral B-cell lymphomas. The CP lymphomas generally appear to arise, even in mice subjected to immunization protocols to activate germinal center reaction, from naive B cells that had not undergone immunoglobulin (Ig) heavy chain gene class switching or somatic hypermutation. In contrast to thymic lymphomas that arise in p53-deficient mice, which generally lack clonal translocations, nearly all analyzed CP B-cell tumors carried clonal translocations. However, in contrast to spontaneous translocations in other mouse B-cell tumor models, CP B-cell tumor translocations were not recurrent and did not involve Ig loci. Therefore, CP tumors might provide models for human lymphomas lacking Ig translocations, such as splenic marginal zone B-cell lymphoma or Waldenstrom macroglobulinemia. Our studies indicate that deletion of p53 is sufficient to trigger transformation of mature B cells and support the notion that p53 deficiency may allow accumulation of oncogenic translocations in B cells.

The ataxia telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements.

Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre-B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.

Novel mechanism of tumor suppression by polarity gene discs large 1 (DLG1) revealed in a murine model of pediatric B-ALL.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel "pre-leukemic" stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers.